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Abstract:

The present technology provides for a microfluidic substrate configured to
carry out PCR on a number of polynucleotide-containing samples in
parallel. The substrate can be a single-layer substrate in a microfluidic
cartridge. Also provided are a method of making a microfluidic cartridge
comprising such a substrate. Still further disclosed are a microfluidic
valve suitable for use in isolating a PCR chamber in a microfluidic
substrate, and a method of making such a valve.

Claims:

1. A microfluidic substrate, comprising:a first PCR reaction chamber;a
second PCR reaction chamber;a first inlet, in fluid communication with
the first PCR reaction chamber;a second inlet, in fluid communication
with the second PCR reaction chamber;a first set of microfluidic valves
configured to isolate the first reaction chamber from the first inlet;
anda second set of microfluidic valves configured to isolate the second
PCR reaction chamber from the second inlet.

2. A microfluidic substrate, comprising:a plurality of sample lanes,
wherein each of the plurality of sample lanes comprises a microfluidic
network having, in fluid communication with one another:an inlet;a first
valve and a second valve;a first channel leading from the inlet, via the
first valve, to a reaction chamber; anda second channel leading from the
reaction chamber, via the second valve, to a vent.

3. The microfluidic substrate of claim 2, additionally comprising:a third
channel leading from the inlet to the reaction chamber, wherein a gate is
positioned in the third channel, and wherein the gate is configured to
open the third channel to permit material from the reaction chamber to be
removed from the cartridge via the inlet.

4. The microfluidic substrate of claim 2, wherein each of the plurality of
sample lanes is configured to amplify one or more polynucleotides
independently of the other lanes.

5. The microfluidic substrate of claim 2, wherein each of the plurality of
sample lanes further comprises a bubble vent.

6. The microfluidic substrate of claim 2, wherein the inlet is configured
to accept sample from a pipette tip.

7. The microfluidic substrate of claim 2, configured to carry out
real-time PCR in at least one of the reaction chambers.

8. The microfluidic substrate of claim 2, wherein the inlets of the
respective plurality of sample lanes are spaced apart from one another to
permit simultaneous loading from a multiple-pipette head dispenser.

9. The microfluidic substrate of claim 2, wherein the first and second
valves comprise a temperature responsive substance that melts upon
heating and seals the reaction chamber.

11. The microfluidic cartridge of claim 10, further comprising a
registration member that ensures that the cartridge is received by a
complementary diagnostic apparatus in a single orientation.

12. The microfluidic cartridge of claim 10, wherein each of the
microfluidic networks, including the PCR reaction chamber, the inlet hole
and the valves for isolating the PCR reaction chamber, is defined in a
single substrate.

13. The microfluidic cartridge, of claim 12, wherein the substrate is a
rigid substrate and impervious to air or liquid, and entry or exit of air
or liquid during operation of the cartridge is only possible through the
inlet or a vent.

14. A method of carrying out PCR independently on a plurality of
polynucleotide-containing samples, the method comprising:introducing the
plurality of samples in to a microfluidic cartridge, wherein the
cartridge has a plurality of PCR reaction chambers configured to permit
thermal cycling of the plurality of samples independently of one
another;moving the plurality of samples into the respective plurality of
PCR reaction chambers;isolating the plurality of PCR reaction chambers;
andamplifying polynucleotides contained with the plurality of samples, by
application of successive heating and cooling cycles to the PCR reaction
chambers.

15. A microfluidic valve, comprising:a first chamber, connected to a first
load channel;a second chamber, connected to a second load channel; anda
flow channel,wherein the first and second load channels are each
connected to the flow channel, andwherein the first and second load
channels each contain a thermally responsive substance that, upon
actuation of the valve, flows into the flow channel thereby sealing it,
andwherein the flow channel is constricted along a length either side of
the first and second load channels.

16. The valve of claim 14, wherein the constricted portion of the flow
channel additionally comprises at least one curve.

17. A microfluidic valve, comprising:a chamber, connected to a load
channel; anda flow channel,wherein the load channel is connected to the
flow channel, andwherein the load channel contains a thermally responsive
substance that, upon actuation of the valve, flows into the flow channel
thereby sealing it, andwherein the flow channel is constricted along a
length either side of the load channel.

Description:

CLAIM OF PRIORITY

[0001]The instant application claims the benefit of priority to U.S.
provisional applications having Ser. Nos. 60/859,284, filed Nov. 14,
2006, and 60/959,437, filed Jul. 13, 2007, the specifications of both of
which are incorporated herein by reference in their entireties.

TECHNICAL FIELD

[0002]The technology described herein generally relates to microfluidic
cartridges. The technology more particularly relates to microfluidic
cartridges that are configured to carry out PCR on nucleotides of
interest, particularly from several biological samples in parallel,
within microfluidic channels in the cartridge, and permit detection of
those nucleotides.

BACKGROUND

[0003]The medical diagnostics industry is a critical element of today's
healthcare infrastructure. At present, however, diagnostic analyses no
matter how routine have become a bottleneck in patient care. There are
several reasons for this. First, many diagnostic analyses can only be
done with highly specialist equipment that is both expensive and only
operable by trained clinicians. Such equipment is found in only a few
locations--often just one in any given urban area. This means that most
hospitals are required to send out samples for analyses to these
locations, thereby incurring shipping costs and transportation delays,
and possibly even sample loss or mix-up. Second, the equipment in
question is typically not available `on-demand` but instead runs in
batches, thereby delaying the processing time for many samples because
they must wait for a machine to fill up before they can be run.

[0004]Understanding that sample flow breaks down into several key steps,
it would be desirable to consider ways to automate as many of these as
possible. For example, a biological sample, once extracted from a
patient, must be put in a form suitable for a processing regime that
typically involves using polymerase chain reaction (PCR) to amplify a
vector of interest. Once amplified, the presence or absence of a
nucleotide of interest from the sample needs to be determined
unambiguously. Sample preparation is a process that is susceptible to
automation but is also relatively routinely carried out in almost any
location. By contrast, steps such as PCR and nucleotide detection have
customarily only been within the compass of specially trained individuals
having access to specialist equipment.

[0005]There is therefore a need for a method and apparatus of carrying out
PCR on prepared biological samples and detecting amplified nucleotides,
preferably with high throughput. In particular there is a need for an
easy-to-use device that can deliver a diagnostic result on several
samples in a short time.

[0006]The discussion of the background to the technology herein is
included to explain the context of the technology. This is not to be
taken as an admission that any of the material referred to was published,
known, or part of the common general knowledge as at the priority date of
any of the claims.

[0007]Throughout the description and claims of the specification the word
"comprise" and variations thereof, such as "comprising" and "comprises",
is not intended to exclude other additives, components, integers or
steps.

SUMMARY

[0008]The present technology includes methods and devices for detecting
polynucleotides in samples, particularly from biological samples. In
particular, the technology relates to microfluidic devices that carry out
PCR on nucleotides of interest within microfluidic channels, and permit
detection of those nucleotides.

[0009]In particular, the present technology provides for a microfluidic
cartridge, comprising: a first PCR reaction chamber; a second PCR
reaction chamber; a first inlet, in fluid communication with the first
PCR reaction chamber; a second inlet, in fluid communication with the
second PCR reaction chamber; a first set of microfluidic valves
configured to control motion of a sample from the first inlet to the
first PCR reaction chamber; and a second set of microfluidic valves
configured to control motion of a sample from the second inlet to the
second PCR reaction chamber.

[0010]The present technology includes a process for carrying out PCR on a
plurality of polynucleotide-containing samples, the method comprising:
introducing the plurality of samples into a microfluidic cartridge,
wherein the cartridge has a plurality of PCR reaction chambers configured
to permit thermal cycling of the plurality of samples independently of
one another; moving the plurality of samples into the respective
plurality of PCR reaction chambers; and amplifying polynucleotides
contained with the plurality of samples, by application of successive
heating and cooling cycles to the PCR reaction chambers.

[0011]The present technology further comprises a number of other
embodiments, as set forth herein.

[0012]A microfluidic substrate, comprising: a first PCR reaction chamber;
a second PCR reaction chamber; a first inlet, in fluid communication with
the first PCR reaction chamber; a second inlet, in fluid communication
with the second PCR reaction chamber; a first set of microfluidic valves
configured to isolate the first reaction chamber from the first inlet;
and a second set of microfluidic valves configured to isolate the second
PCR reaction chamber from the second inlet.

[0013]A microfluidic substrate, comprising: a plurality of sample lanes,
wherein each of the plurality of sample lanes comprises a microfluidic
network having, in fluid communication with one another: an inlet; a
first valve and a second valve; a first channel leading from the inlet,
via the first valve, to a reaction chamber; and a second channel leading
from the reaction chamber, via the second valve, to a vent.

[0014]A microfluidic cartridge having a plurality of microfluidic
networks, wherein each of the microfluidic networks, including a PCR
reaction chamber, an inlet hole, and the valves for isolating the PCR
reaction chamber, is defined in a single substrate.

[0015]A method of carrying out PCR independently on a plurality of
polynucleotide-containing samples, the method comprising: introducing the
plurality of samples in to a microfluidic cartridge, wherein the
cartridge has a plurality of PCR reaction chambers configured to permit
thermal cycling of the plurality of samples independently of one another;
moving the plurality of samples into the respective plurality of PCR
reaction chambers; isolating the plurality of PCR reaction chambers; and
amplifying polynucleotides contained with the plurality of samples, by
application of successive heating and cooling cycles to the PCR reaction
chambers.

[0016]A microfluidic valve, comprising: a first chamber, connected to a
first load channel; a second chamber, connected to a second load channel;
and a flow channel, wherein the first and second load channels are each
connected to the flow channel, and wherein the first and second load
channels each contain a thermally responsive substance that, upon
actuation of the valve, flows into the flow channel thereby sealing it,
and wherein the flow channel is constricted along a length either side of
the first and second load channels.

[0017]A microfluidic valve, comprising: a chamber, connected to a load
channel; and a flow channel, wherein the load channel is connected to the
flow channel, and wherein the load channel contains a thermally
responsive substance that, upon actuation of the valve, flows into the
flow channel thereby sealing it, and wherein the flow channel is
constricted along a length either side of the load channel.

[0018]A method of making a microfluidic valve, the method comprising:
directing a dispensing head over an inlet hole in a microfluidic
substrate; propelling a quantity of thermally responsive substance from
the dispensing head into the inlet hole; and maintaining a temperature of
the microfluidic substrate so that the thermally responsive substance
flows by capillary action from the inlet hole into a microfluidic channel
in communication with the inlet hole.

[0019]The microfluidic cartridge described herein can be configured for
use with an apparatus comprising: a chamber configured to receive the
microfluidic cartridge; at least one heat source thermally coupled to the
cartridge and configured to apply heat and cooling cycles that carry out
PCR on one or more microdroplets of polynucleotide-containing sample in
the cartridge; a detector configured to detect presence of one or more
polynucleotides in the one or more samples; and a processor coupled to
the detector and the heat source, configured to control heating of one or
more regions of the microfluidic cartridge.

[0020]The details of one or more embodiments of the technology are set
forth in the accompanying drawings and further description herein. Other
features, objects, and advantages of the technology will be apparent from
the description and drawings, and from the claims.

[0032]FIG. 14 shows a cross-section of a portion of a microfluidic
cartridge, when in contact with a heater substrate;

[0033]FIGS. 15A and 15B show a plan view of heater circuitry adjacent to a
PCR reaction chamber; FIG. 15c shows an overlay of an array of heater
elements on an exemplary multi-lane microfluidic cartridge, wherein
various microfluidic networks are visible;

[0034]FIG. 16 shows various cut-away sections that can be used to improve
cooling rates during PCR thermal cycling;

[0035]FIG. 17 shows a plot of temperature against time during a PCR
process, as performed on a microfluidic cartridge as described herein;

[0036]FIG. 18 shows an exemplary assembly process for a cartridge as
further described herein;

[0037]FIGS. 19A and 19B show exemplary deposition of wax droplets into
microfluidic valves;

[0038]FIG. 20 shows an exemplary apparatus, a microfluidic cartridge, and
a read head contains a detector, as further described herein;

[0039]FIG. 21 shows a cross-section of a pipetting head and a cartridge in
position in a microfluidic apparatus;

[0040]FIG. 22 shows introduction of a PCR-ready sample into a cartridge,
situated in an instrument;

[0050]The present technology comprises a microfluidic cartridge that is
configured to carry out an amplification, such as by PCR, of one or more
polynucleotides from one or more samples. It is to be understood that,
unless specifically made clear to the contrary, where the term PCR is
used herein, any variant of PCR including but not limited to real-time
and quantitative, and any other form of polynucleotide amplification is
intended to be encompassed. The microfluidic cartridge need not be
self-contained and can be designed so that it receives thermal energy
from one or more heating elements present in an external apparatus with
which the cartridge is in thermal communication. An exemplary such
apparatus is further described herein; additional embodiments of such a
system are found in U.S. patent application Ser. No. ______, entitled
"Microfluidic System for Amplifying and Detecting Polynucleotides in
Parallel", and filed on even date herewith, the specification of which is
incorporated herein by reference.

[0051]By cartridge is meant a unit that may be disposable, or reusable in
whole or in part, and that is configured to be used in conjunction with
some other apparatus that has been suitably and complementarily
configured to receive and operate on (such as deliver energy to) the
cartridge.

[0052]By microfluidic, as used herein, is meant that volumes of sample,
and/or reagent, and/or amplified polynucleotide are from about 0.1 μl
to about 999 μl, such as from 1-100 μl, or from 2-25 μl.
Similarly, as applied to a cartridge, the term microfluidic means that
various components and channels of the cartridge, as further described
herein, are configured to accept, and/or retain, and/or facilitate
passage of microfluidic volumes of sample, reagent, or amplified
polynucleotide. Certain embodiments herein can also function with
nanoliter volumes (in the range of 10-500 nanoliters, such as 100
nanoliters).

[0053]One aspect of the present technology relates to a microfluidic
cartridge having two or more sample lanes arranged so that analyses can
be carried out in two or more of the lanes in parallel, for example
simultaneously, and wherein each lane is independently associated with a
given sample.

[0054]A sample lane is an independently controllable set of elements by
which a sample can be analyzed, according to methods described herein as
well as others known in the art. A sample lane comprises at least a
sample inlet, and a microfluidic network having one or more microfluidic
components, as further described herein.

[0055]In various embodiments, a sample lane can include a sample inlet
port or valve, and a microfluidic network that comprises, in fluidic
communication one or more components selected from the group consisting
of: at least one thermally actuated valve, a bubble removal vent, at
least one thermally actuated pump, a gate, mixing channel, positioning
element, microreactor, a downstream thermally actuated valve, and a PCR
reaction chamber. The sample inlet valve can be configured to accept a
sample at a pressure differential compared to ambient pressure of between
about 70 and 100 kilopascals.

[0056]The cartridge can therefore include a plurality of microfluidic
networks, each network having various components, and each network
configured to carry out PCR on a sample in which the presence or absence
of one or more polynucleotides is to be determined.

[0057]A multi-lane cartridge is configured to accept a number of samples
in series or in parallel, simultaneously or consecutively, in particular
embodiments 12 samples, wherein the samples include at least a first
sample and a second sample, wherein the first sample and the second
sample each contain one or more polynucleotides in a form suitable for
amplification. The polynucleotides in question may be the same as, or
different from one another, in different samples and hence in different
lanes of the cartridge. The cartridge typically processes each sample by
increasing the concentration of a polynucleotide to be determined and/or
by reducing the concentration of inhibitors relative to the concentration
of polynucleotide to be determined.

[0058]The multi-lane cartridge comprises at least a first sample lane
having a first microfluidic network and a second lane having a second
microfluidic network, wherein each of the first microfluidic network and
the second microfluidic network is as elsewhere described herein, and
wherein the first microfluidic network is configured to amplify
polynucleotides in the first sample, and wherein the second microfluidic
network is configured to amplify polynucleotides in the second sample.

[0059]In various embodiments, the microfluidic network can be configured
to couple heat from an external heat source to a sample mixture
comprising PCR reagent and neutralized polynucleotide sample under
thermal cycling conditions suitable for creating PCR amplicons from the
neutralized polynucleotide sample.

[0060]At least the external heat source may operate under control of a
computer processor, configured to execute computer readable instructions
for operating one or more components of each sample lane, independently
of one another, and for receiving signals from a detector that measures
fluorescence from one or more of the PCR reaction chambers.

[0061]For example, FIG. 1 shows a plan view of a microfluidic cartridge
100 containing twelve independent sample lanes 101 capable of
simultaneous or successive processing. The microfluidic network in each
lane is typically configured to carry out amplification, such as by PCR,
on a PCR-ready sample, such as one containing nucleic acid extracted from
a sample using other methods as further described herein. A PCR-ready
sample is thus typically a mixture comprising the PCR reagents and the
neutralized polynucleotide sample, suitable for subjecting to thermal
cycling conditions that create PCR amplicons from the neutralized
polynucleotide sample. For example, a PCR-ready sample can include a PCR
reagent mixture comprising a polymerase enzyme, a positive control
plasmid, a fluorogenic hybridization probe selective for at least a
portion of the plasmid and a plurality of nucleotides, and at least one
probe that is selective for a polynucleotide sequence. Exemplary probes
are further described herein. Typically, the microfluidic network is
configured to couple heat from an external heat source with the mixture
comprising the PCR reagent and the neutralized polynucleotide sample
under thermal cycling conditions suitable for creating PCR amplicons from
the neutralized polynucleotide sample.

[0062]In various embodiments, the PCR reagent mixture can include a
positive control plasmid and a plasmid fluorogenic hybridization probe
selective for at least a portion of the plasmid, and the microfluidic
cartridge can be configured to allow independent optical detection of the
fluorogenic hybridization probe and the plasmid fluorogenic hybridization
probe.

[0063]In various embodiments, the microfluidic cartridge can accommodate a
negative control polynucleotide, wherein the microfluidic network can be
configured to independently carry out PCR on each of a neutralized
polynucleotide sample and a negative control polynucleotide with the PCR
reagent mixture under thermal cycling conditions suitable for
independently creating PCR amplicons of the neutralized polynucleotide
sample and PCR amplicons of the negative control polynucleotide. Each
lane of a multi-lane cartridge as described herein can perform two
reactions when used in conjunction with two fluorescence detection
systems per lane. A variety of combinations of reactions can be performed
in the cartridge, such as two sample reactions in one lane, a positive
control and a negative control in two other lanes; or a sample reaction
and an internal control in one lane and a negative control in a separate
lane.

[0064]FIG. 2A shows a perspective view of a portion of an exemplary
microfluidic cartridge 200 according to the present technology. FIG. 2B
shows a close-up view of a portion of the cartridge 200 of FIG. 2A
illustrating various representative components. The cartridge 200 may be
referred to as a multi-lane PCR cartridge with dedicated sample inlets
202. For example sample inlet 202 is configured to accept a liquid
transfer member (not shown) such as a syringe, a pipette, or a PCR tube
containing a PCR ready sample. More than one inlet 202 is shown in FIGS.
2A, 2B, wherein one inlet operates in conjunction with a single sample
lane. Various components of microfluidic circuitry in each lane are also
visible. For example, microvalves 204, and 206, and hydrophobic vents 208
for removing air bubbles, are parts of microfluidic circuitry in a given
lane. Also shown is an ultrafast PCR reactor 210, which, as further
described herein, is a microfluidic channel in a given sample lane that
is long enough to permit PCR to amplify polynucleotides present in a
sample. Above each PCR reactor 210 is a window 212 that permits detection
of fluorescence from a fluorescent substance in PCR reactor 210 when a
detector is situated above window 212. It is to be understood that other
configurations of windows are possible including, but not limited to, a
single window that straddles each PCR reactor across the width of
cartridge 200.

[0065]In preferred embodiments, the multi-sample cartridge has a size
substantially the same as that of a 96-well plate as is customarily used
in the art. Advantageously, then, such a cartridge may be used with plate
handlers used elsewhere in the art.

[0066]The sample inlets of adjacent lanes are reasonably spaced apart from
one another to prevent any contamination of one sample inlet from another
sample when a user introduces a sample into any one cartridge. In an
embodiment, the sample inlets are configured so as to prevent subsequent
inadvertent introduction of sample into a given lane after a sample has
already been introduced into that lane. In certain embodiments, the
multi-sample cartridge is designed so that a spacing between the
centroids of sample inlets is 9 mm, which is an industry-recognized
standard. This means that, in certain embodiments the center-to-center
distance between inlet holes in the cartridge that accept samples from
PCR tubes, as further described herein, is 9 mm. The inlet holes can be
manufactured conical in shape with an appropriate conical angle so that
industry-standard pipette tips (2 μl, 20 μl, 200 μl, volumes,
etc.) fit snugly therein. The cartridge herein may be adapted to suit
other, later-arising, industry standards not otherwise described herein,
as would be understood by one of ordinary skill in the art.

[0067]FIG. 3 shows a plan view of an exemplary microfluidic cartridge 300
having 12 sample lanes. The inlet ports 302 in this embodiment have a 6
mm spacing, so that, when used in conjunction with an automated sample
loader having 4 heads, spaced equidistantly at 18 mm apart, the inlets
can be loaded in three batches of four inlets: e.g., inlets 1, 4, 7, and
10 together, followed by 2, 5, 8, and 11, then finally 3, 6, 9, and 12,
wherein the 12 inlets are numbered consecutively from one side of the
cartridge to the other as shown.

[0068]A microfluidic cartridge as used herein may be constructed from a
number of layers. Accordingly, one aspect of the present technology
relates to a microfluidic cartridge that comprises a first, second,
third, fourth, and fifth layers wherein one or more layers define a
plurality of microfluidic networks, each network having various
components configured to carry out PCR on a sample in which the presence
or absence of one or more polynucleotides is to be determined. In various
embodiments, one or more such layers are optional.

[0069]FIGS. 4A-C show various views of a layer structure of an exemplary
microfluidic cartridge comprising a number of layers, as further
described herein. FIG. 4A shows an exploded view; FIG. 4B shows a
perspective view; and FIG. 4c shows a cross-sectional view of a sample
lane in the exemplary cartridge. Referring to FIGS. 4A-C, an exemplary
microfluidic cartridge 400 includes first 420, second 422, third 424,
fourth 426, and fifth layers in two non-contiguous parts 428, 430 (as
shown) that enclose a microfluidic network having various components
configured to process multiple samples in parallel that include one or
more polynucleotides to be determined.

[0070]Microfluidic cartridge 400 can be fabricated as desired. The
cartridge can include a microfluidic substrate layer 424, typically
injection molded out of a plastic, such as a zeonor plastic (cyclic
olefin polymer), having a PCR channel and valve channels on a first side
and vent channels and various inlet holes, including wax loading holes
and liquid inlet holes, on a second side (disposed toward hydrophobic
vent membrane 426). It is advantageous that all the microfluidic network
defining structures, such as PCR reactors, valves, inlet holes, and air
vents, are defined on the same single substrate 424. This attribute
facilitates manufacture and assembly of the cartridge. Additionally, the
material from which this substrate is formed is rigid or non-deformable,
non-venting to air and other gases, and has a low autofluorescence to
facilitate detection of polynucleotides during an amplification reaction
performed in the microfluidic circuitry defined therein. Rigidity is
advantageous because it facilitates effective and uniform contact with a
heat unit as further described herein. Use of a non-venting material is
also advantageous because it reduces the likelihood that the
concentration of various species in liquid form will change during
analysis. Use of a material having low auto-fluorescence is also
important so that background fluorescence does not detract from
measurement of fluorescence from the analyte of interest.

[0071]The cartridge can further include, disposed on top of the substrate
424, an oleophobic/hydrophobic vent membrane layer 426 of a porous
material, such as 0.2 to 1.0 micron pore-size membrane of modified
polytetrafluorethylene, the membrane being typically between about 25 and
about 100 microns thick, and configured to cover the vent channels of
microfluidic substrate 424, and attached thereto using, for example, heat
bonding.

[0072]Typically, the microfluidic cartridge further includes a layer 428,
430 of polypropylene or other plastic label with pressure sensitive
adhesive (typically between about 50 and 150 microns thick) configured to
seal the wax loading holes of the valves in substrate 424, trap air used
for valve actuation, and serve as a location for operator markings. In
FIG. 4A, this layer is shown in two separate pieces, 228, 230, though it
would be understood by one of ordinary skill in the art that a single
piece layer would be appropriate.

[0073]In various embodiments, the label is a computer-readable label. For
example, the label can include a bar code, a radio frequency tag or one
or more computer-readable characters. The label can be formed of a
mechanically compliant material. For example, the mechanically compliant
material of the label can have a thickness of between about 0.05 and
about 2 millimeters and a Shore hardness of between about 25 and about
100. The label can be positioned such that it can be read by a sample
identification verifier as further described herein.

[0074]The cartridge can further include a heat sealable laminate layer 422
(typically between about 100 and about 125 microns thick) attached to the
bottom surface of the microfluidic substrate 424 using, for example, heat
bonding. This layer serves to seal the PCR channels and vent channels in
substrate 424. The cartridge can further include a thermal interface
material layer 420 (typically about 125 microns thick), attached to the
bottom of the heat sealable laminate layer using, for example, pressure
sensitive adhesive. The layer 420 can be compressible and have a higher
thermal conductivity than common plastics, thereby serving to transfer
heat across the laminate more efficiently. Typically, however, layer 420
is not present.

[0075]The application of pressure to contact the cartridge to the heater
of an instrument that receives the cartridge generally assists in
achieving better thermal contact between the heater and the
heat-receivable parts of the cartridge, and also prevents the bottom
laminate structure from expanding, as would happen if the PCR channel was
only partially filled with liquid and the air entrapped therein would be
thermally expanded during thermocycling.

[0076]In use, cartridge 400 is typically thermally associated with an
array of heat sources configured to operate the components (e.g., valves,
gates, actuators, and processing region 410) of the device. Exemplary
such heater arrays are further described herein. Additional embodiments
of heater arrays are described in U.S. patent application Ser. No.
______, entitled "Heater Unit for Microfluidic Diagnostic System" and
filed on even date herewith, the specification of which is incorporated
herein by reference in its entirety. In some embodiments, the heat
sources are controlled by a computer processor and actuated according to
a desired protocol. Processors configured to operate microfluidic devices
are described in, e.g., U.S. application Ser. No. 09/819,105, filed Mar.
28, 2001, which application is incorporated herein by reference.

[0077]In various embodiments, during transport and storage, the
microfluidic cartridge can be further surrounded by a sealed pouch. The
microfluidic cartridge can be sealed in the pouch with an inert gas. The
microfluidic cartridge can be disposable for example after one or more of
its sample lanes have been used.

Highly Multiplexed Embodiments

[0078]Embodiments of the cartridge described herein may be constructed
that have high-density microfluidic circuitry on a single cartridge that
thereby permit processing of multiple samples in parallel, or in
sequence, on a single cartridge. Preferred numbers of such multiple
samples include 20, 24, 36, 40, 48, 50, 60, 64, 72, 80, 84, 96, and 100,
but it would be understood that still other numbers are consistent with
the apparatus and cartridge herein, where deemed convenient and
practical.

[0079]Accordingly, different configurations of lanes, sample inlets, and
associated heater networks than those explicitly depicted in the FIGs and
examples that can facilitate processing such numbers of samples on a
single cartridge are within the scope of the instant disclosure.
Similarly, alternative configurations of detectors and heating elements
for use in conjunction with such a highly multiplexed cartridge are also
within the scope of the description herein.

[0080]It is also to be understood that the microfluidic cartridges
described herein are not to be limited to rectangular shapes, but can
include cartridges having circular, elliptical, triangular, rhombohedral,
square, and other shapes. Such shapes may also be adapted to include some
irregularity, such as a cut-out, to facilitate placement in a
complementary apparatus as further described herein.

[0081]In an exemplary embodiment, a highly multiplexed cartridge has 48
sample lanes, and permits independent control of each valve in each lane
by suitably configured heater circuitry, with 2 banks of thermocycling
protocols per lane, as shown in FIG. 5. In the embodiment in FIG. 5, the
heaters (shown superimposed on the lanes) are arranged in three arrays
502, 504, with 506, and 508. The heaters are themselves disposed within
one or more substrates. Heater arrays 502, 508 in two separate glass
regions only apply heat to valves in the microfluidic networks in each
lane. Because of the low thermal conductivity of glass, the individual
valves may be heated separately from one another. This permits samples to
be loaded into the cartridge at different times, and passed to the PCR
reaction chambers independently of one another. The PCR heaters 504, 506
are mounted on a silicon substrate--and are not readily heated
individually, but thereby permit batch processing of PCR samples, where
multiple samples from different lanes are amplified by the same set of
heating/cooling cycles. It is preferable for the PCR heaters to be
arranged in 2 banks (the heater arrays 506 on the left and right 508 are
not in electrical communication with one another), thereby permitting a
separate degree of sample control.

[0082]FIG. 6 shows a representative 48-sample cartridge 600 compatible
with the heater arrays of FIG. 5, and having a configuration of inlets
602 different to that depicted o other cartridges herein. The inlet
configuration is exemplary and has been designed to maximize efficiency
of space usage on the cartridge. The inlet configuration can be
compatible with an automatic pipetting machine that has dispensing heads
situated at a 9 mm spacing. For example, such a machine having 4 heads
can load 4 inlets at once, in 12 discrete steps, for the cartridge of
FIG. 6. Other configurations of inlets though not explicitly described or
depicted are compatible with the technology described herein.

[0083]FIG. 7 shows, in close up, an exemplary spacing of valves 702,
channels 704, and vents 796, in adjacent lanes 708 of a multi-sample
microfluidic cartridge for example as shown in FIG. 6.

[0085]FIGS. 10A and 10B show various views of an embodiment of a
radially-configured highly-multiplexed cartridge, having a number of
inlets 1002, microfluidic lanes 1004, valves 1005, and PCR reaction
chambers 1006. FIG. 10c shows an array of heater elements 1008 compatible
with the cartridge layout of FIG. 10A.

[0086]The various embodiments shown in FIGS. 5-10C are compatible with
liquid dispensers, receiving bays, and detectors that are configured
differently from the other specific examples described herein.

[0087]During the design and manufacture of highly multiplexed cartridges,
photolithographic processing steps such as etching, hole
drilling/photo-chemical drilling/sand-blasting/ion-milling processes
should be optimized to give well defined holes and microchannel pattern.
Proper distances between channels should be identified and maintained to
obtain good bonding between the microchannel substrate and the heat
conducting substrate layer. In particular, it is desirable that minimal
distances are maintained between pairs of adjacent microchannels to
promote, reliable bonding of the laminate in between the channels.

[0088]The fabrication by injection molding of these complicated
microfluidic structures having multiple channels and multiple inlet holes
entails proper consideration of dimensional repeatability of these
structures over multiple shots from the injection molding master pattern.
Proper consideration is also attached to the placement of ejector pins to
push out the structure from the mold without causing warp, bend or
stretching of it. For example, impression of the ejector pins on the
microfluidic substrate should not sink into the substrate thereby
preventing planarity of the surface of the cartridge. The accurate
placement of various inlet holes (such as sample inlet holes, valve inlet
holes and vent holes) relative to adjacent microfluidic channels is also
important because the presence of these holes can cause knit-lines to
form that might cause unintended leak from a hole to a microchannel.
Highly multiplexed microfluidic substrates may be fabricated in other
materials such as glass, silicon.

[0089]The size of the substrate relative to the number of holes is also
factor during fabrication because it is easy to make a substrate having
just a simple microfluidic network with a few holes (maybe fewer than 10
holes) and a few microchannels, but making a substrate having over 24, or
over 48, or over 72 holes, etc., is more difficult.

Microfluidic Networks

[0090]Particular components of exemplary microfluidic networks are further
described herein.

[0091]Channels of a microfluidic network in a lane of cartridge typically
have at least one sub-millimeter cross-sectional dimension. For example,
channels of such a network may have a width and/or a depth of about 1 mm
or less (e.g., about 750 microns or less, about 500 microns, or less,
about 250 microns or less).

[0092]FIG. 11 shows a plan view of a representative microfluidic circuit
found in one lane of a multi-lane cartridge such as shown in FIGS. 2A and
2B. It would be understood by one skilled in the art that other
configurations of microfluidic network would be consistent with the
function of the cartridges and apparatus described herein. In operation
of the cartridge, in sequence, sample is introduced through liquid inlet
202, optionally flows into a bubble removal vent channel 208 (which
permits adventitious air bubbles introduced into the sample during entry,
to escape), and continues along a channel 216. Typically, when using a
robotic dispenser of liquid sample, the volume is dispensed accurately
enough that formation of bubbles is not a significant problem, and the
presence of vent channel 208 is not necessary. Thus, in certain
embodiments, the bubble removal vent channel 208 is not present and
sample flows directly into channel 216. Throughout the operation of
cartridge 200, the fluid is manipulated as a microdroplet (not shown in
FIG. 11). Valves 204 and 206 are initially both open, so that a
microdroplet of sample-containing fluid can be pumped into PCR reactor
channel 210 from inlet hole 202 under influence of force from the sample
injection operation. Upon initiating of processing, the detector present
on top of the PCR reactor 210 checks for the presence of liquid in the
PCR channel, and then valves 204 and 206 are closed to isolate the PCR
reaction mix from the outside. In one embodiment, the checking of the
presence of liquid in the PCR channel is by measuring the heat ramp rate,
such as by one or more temperature sensors in the heating unit. A channel
with liquid absent will heat up faster than one in which, e.g., a sample,
is present.

[0093]Both valves 204 and 206 are closed prior to thermocycling to prevent
or reduce any evaporation of liquid, bubble generation, or movement of
fluid from the PCR reactor. End vent 214 is configured to prevent a user
from introducing an excess amount of liquid into the microfluidic
cartridge, as well as playing a role of containing any sample from
spilling over to unintended parts of the cartridge. A user may input
sample volumes as small as an amount to fill the region from the bubble
removal vent (if present) to the middle of the microreactor, or up to
valve 204 or beyond valve 204. The use of microvalves prevents both loss
of liquid or vapor thereby enabling even a partially filled reactor to
successfully complete a PCR thermocycling reaction.

[0094]The reactor 210 is a microfluidic channel that is heated through a
series of cycles to carry out amplification of nucleotides in the sample,
as further described herein, and according to amplification protocols
known to those of ordinary skill in the art. The inside walls of the
channel in the PCR reactor are typically made very smooth and polished to
a shiny finish (for example, using a polish selected from SPI A1, SPI A2,
SPI A3, SPI B1, or SPI B2) during manufacture. This is in order to
minimize any microscopic quantities of air trapped in the surface of the
PCR channel, which would causing bubbling during the thermocycling steps.
The presence of bubbles especially in the detection region of the PCR
channel could also cause a false or inaccurate reading while monitoring
progress of the PCR. Additionally, the PCR channel can be made shallow
such that the temperature gradient across the depth of the channel is
minimized.

[0095]The region of the cartridge 212 above PCR reactor 210 is a thinned
down section to reduce thermal mass and autofluorescence from plastic in
the cartridge. It permits a detector to more reliably monitor progress of
the reaction and also to detect fluorescence from a probe that binds to a
quantity of amplified nucleotide. Exemplary probes are further described
herein. The region 212 can be made of thinner material than the rest of
the cartridge so as to permit the PCR channel to be more responsive to a
heating cycle (for example, to rapidly heat and cool between temperatures
appropriate for denaturing and annealing steps), and so as to reduce
glare, autofluorescence, and undue absorption of fluorescence.

[0096]After PCR has been carried out on a sample, and presence or absence
of a polynucleotide of interest has been determined, it is preferred that
the amplified sample remains in the cartridge and that the cartridge is
either used again (if one or more lanes remain unused), or disposed of.
Should a user wish to run a post amplification analysis, such as gel
electrophoresis, the user may pierce a hole through the laminate of the
cartridge, and recover an amount--typically about 1.5 microliter--of PCR
product. The user may also place the individual PCR lane on a special
narrow heated plate, maintained at a temperature to melt the wax in the
valve, and then aspirate the reacted sample from the inlet hole of that
PCR lane.

[0097]In various embodiments, the microfluidic network can optionally
include at least one reservoir configured to contain waste.

[0098]Table 1 outlines typical volumes, pumping pressures, and operation
times associated with various components of a microfluidic cartridge
described herein.

[0099]A valve (sometimes referred to herein as a microvalve) is a
component in communication with a channel, such that the valve has a
normally open state allowing material to pass along a channel from a
position on one side of the valve (e.g., upstream of the valve) to a
position on the other side of the valve (e.g., downstream of the valve).
Upon actuation of the valve, the valve transitions to a closed state that
prevents material from passing along the channel from one side of the
valve to the other. For example, in one embodiment, a valve can include a
mass of a thermally responsive substance (TRS) that is relatively
immobile at a first temperature and more mobile at a second temperature.
The first and second temperatures are insufficiently high to damage
materials, such as polymer layers of a microfluidic cartridge in which
the valve is situated. A mass of TRS can be an essentially solid mass or
an agglomeration of smaller particles that cooperate to obstruct the
passage when the valve is closed. Examples of TRS's include a eutectic
alloy (e.g., a solder), wax (e.g., an olefin), polymers, plastics, and
combinations thereof. The TRS can also be a blend of variety of
materials, such as an emulsion of thermoelastic polymer blended with air
microbubbles (to enable higher thermal expansion, as well as reversible
expansion and contraction), polymer blended with expancel material
(offering higher thermal expansion), polymer blended with heat conducting
microspheres (offering faster heat conduction and hence, faster melting
profiles), or a polymer blended with magnetic microspheres (to permit
magnetic actuation of the melted thermoresponsive material).

[0100]Generally, for such a valve, the second temperature is less than
about 90° C. and the first temperature is less than the second
temperature (e.g., about 70° C. or less). Typically, a chamber is
in gaseous communication with the mass of TRS. The valve is in
communication with a source of heat that can be selectively applied to
the chamber of air and to the TRS. Upon heating gas (e.g., air) in the
chamber and heating the mass of TRS to the second temperature, gas
pressure within the chamber due to expansion of the volume of gas, forces
the mass to move into the channel, thereby obstructing material from
passing therealong.

[0101]An exemplary valve is shown in FIG. 12A. The valve of FIG. 12A has
two chambers of air 1203, 1205 in contact with, respectively, each of two
channels 1207, 1208 containing TRS. The air chambers also serve as
loading ports for TRS during manufacture of the valve, as further
described herein. In order to make the valve sealing very robust and
reliable, the flow channel 1201 (along which, e.g., sample passes) at the
valve junction is made narrow (typically 150 μm wide, and 150 μm
deep or narrower), and the constricted portion of the flow channel is
made at least 0.5 or 1 mm long such that the TRS seals up a long narrow
channel thereby reducing any leakage through the walls of the channel. In
the case of a bad seal, there may be leakage of fluid around walls of
channel, past the TRS, when the valve is in the closed state. In order to
minimize this, the flow channel is narrowed and elongated as much as
possible. In order to accommodate such a length of channel on a cartridge
where space may be at a premium, the flow channel can incorporate one or
more curves 1209 as shown in FIG. 12A. The valve operates by heating air
in the TRS-loading port, which forces the TRS forwards into the
flow-channel in a manner so that it does not come back to its original
position. In this way, both air and TRS are heated during operation.

[0102]In various other embodiments, a valve for use with a microfluidic
network in a microfluidic cartridge herein can be a bent valve as shown
in FIG. 12B. Such a configuration reduces the footprint of the valve and
hence reduces cost per part for highly dense microfluidic cartridges. A
single valve loading hole 1211 is positioned in the center, that serves
as an inlet for thermally responsive substance. The leftmost vent 1213
can be configured to be an inlet for, e.g., sample, and the rightmost
vent 1215 acts as an exit for, e.g., air. This configuration can be used
as a prototype for testing such attributes as valve and channel geometry
and materials.

[0103]In various other embodiments, a valve for use with a microfluidic
network can include a curved valve as shown in FIG. 12c, in order to
reduce the effective cross-section of the valve, thereby enabling
manufacture of cheaper dense microfluidic devices. Such a valve can
function with a single valve loading hole and air chamber 1221 instead of
a pair as shown in FIG. 12A.

Gates

[0104]FIG. 12D shows an exemplary gate as may optionally be used in a
microfluidic network herein. A gate can be a component that can have a
closed state that does not allow material to pass along a channel from a
position on one side of the gate to another side of the gate, and an open
state that does allow material to pass along a channel from a position on
one side of the gate to another side of the gate. Actuation of an open
gate can transition the gate to a closed state in which material is not
permitted to pass from one side of the gate (e.g., upstream of the gate)
to the other side of the gate (e.g., downstream of the gate). Upon
actuation, a closed gate can transition to an open state in which
material is permitted to pass from one side of the gate (e.g., upstream
of the gate) to the other side of the gate (e.g., downstream of the
gate).

[0105]In various embodiments, a microfluidic network can include a narrow
gate 380 as shown in FIG. 12D where a gate loading channel 382 used for
loading wax from a wax loading hole 384 to a gate junction 386 can be
narrower (e.g., approximately 150 μm wide and 100 microns deep). An
upstream channel 388 as well as a downstream channel 390 of the gate
junction 386 can be made wide (e.g., ˜500 μm) and deep (e.g.,
˜500 μm) to help ensure the wax stops at the gate junction 386.
The amount of gate material melted and moved out of the gate junction 386
may be minimized for optimal gate 380 opening. As an off-cartridge heater
may be used to melt the thermally responsive substance in gate 380, a
misalignment of the heater could cause the wax in the gate loading
channel 382 to be melted as well. Therefore, narrowing the dimension of
the loading channel may increase reliability of gate opening. In the case
of excessive amounts of wax melted at the gate junction 386 and gate
loading channel 382, the increased cross-sectional area of the downstream
channel 390 adjacent to the gate junction 386 can prevent wax from
clogging the downstream channel 390 during gate 380 opening. The
dimensions of the upstream channel 388 at the gate junction 386 can be
made similar to the downstream channel 390 to ensure correct wax loading
during gate fabrication.

[0106]In various embodiments, the gate can be configured to minimize the
effective area or footprint of the gate within the network and thus bent
gate configurations, although not shown herein are consistent with the
foregoing description.

Vents

[0107]In various embodiments, the microfluidic network can include at
least one hydrophobic vent in addition to an end vent. A vent is a
general outlet (hole) that may or may not be covered with a hydrophobic
membrane. An exit hole is an example of a vent that need not be covered
by a membrane.

[0108]A hydrophobic vent (e.g., a vent in FIG. 13) is a structure that
permits gas to exit a channel while limiting (e.g., preventing)
quantities of liquid from exiting the channel. Typically, hydrophobic
vents include a layer of porous hydrophobic material (e.g., a porous
filter such as a porous hydrophobic membrane from GE Osmonics,
Minnetonka, Minn.) that defines a wall of the channel. As described
elsewhere herein, hydrophobic vents can be used to position a
microdroplet of sample at a desired location within a microfluidic
network.

[0109]The hydrophobic vents of the present technology are preferably
constructed so that the amount of air that escapes through them is
maximized while minimizing the volume of the channel below the vent
surface. Accordingly, it is preferable that the vent is constructed so as
to have a hydrophobic membrane 1303 of large surface area and a shallow
cross section of the microchannel below the vent surface.

[0110]Hydrophobic vents are useful for bubble removal and typically have a
length of at least about 2.5 mm (e.g., at least about 5 mm, at least
about 7.5 mm) along a channel 1305 (see FIG. 13). The length of the
hydrophobic vent is typically at least about 5 times (e.g., at least
about 10 times, at least about 20 times) larger than a depth of the
channel within the hydrophobic vent. For example, in some embodiments,
the channel depth within the hydrophobic vent is about 300 microns or
less (e.g., about 250 microns or less, about 200 microns or less, about
150 microns or less).

[0111]The depth of the channel within the hydrophobic vent is typically
about 75% or less (e.g., about 65% or less, about 60% or less) of the
depth of the channel upstream 1301 and downstream (not shown) of the
hydrophobic vent. For example, in some embodiments the channel depth
within the hydrophobic vent is about 150 microns and the channel depth
upstream and downstream of the hydrophobic vent is about 250 microns.
Other dimensions are consistent with the description herein.

[0112]A width of the channel within the hydrophobic vent is typically at
least about 25% wider (e.g., at least about 50% wider) than a width of
the channel upstream from the vent and downstream from the vent. For
example, in an exemplary embodiment, the width of the channel within the
hydrophobic vent is about 400 microns, and the width of the channel
upstream and downstream from the vent is about 250 microns. Other
dimensions are consistent with the description herein.

[0113]The vent in FIG. 13 is shown in a linear configuration though it
would be understood that it need not be so. A bent, kinked, curved,
S-shaped, V-shaped, or U-shaped (as in item 208 FIG. 11) vent is also
consistent with the manner of construction and operation described
herein.

Heater Configurations to Ensure Uniform Heating of a Region

[0114]The microfluidic cartridges described herein are configured to
position in a complementary receiving bay in an apparatus that contains a
heater unit. The heater unit is configured to deliver heat to specific
regions of the cartridge, including but not limited to one or more
microfluidic components, at specific times. For example, the heat source
is configured so that particular heating elements are situated adjacent
to specific components of the microfluidic network of the cartridge. In
certain embodiments, the apparatus uniformly controls the heating of a
region of a microfluidic network. In an exemplary embodiment, multiple
heaters can be configured to simultaneously and uniformly heat a single
region, such as the PCR reaction chamber, of the microfluidic cartridge.
In other embodiments, portions of different sample lanes are heated
simultaneously and independently of one another.

[0115]FIG. 14 shows a cross-sectional view of an exemplary microfluidic
cartridge to show the location of a PCR channel in relation to various
heaters when the cartridge is placed in a suitable apparatus. The view in
FIG. 14 is also referred to as a sectional-isometric view of the
cartridge lying over a heater wafer. A window 903 above the PCR channel
in the cartridge is shown in perspective view. PCR channel 901 (for
example, 150μ deep×700μ wide), is shown in an upper layer of
the cartridge. A laminate layer 905 of the cartridge (for example,
125μ thick) is directly under the PCR channel 901. As depicted, an
optional further layer of thermal interface laminate 907 on the cartridge
(for example, 125μ thick) lies directly under the laminate layer 905.
Heaters 909, 911 are situated in a heater substrate layer 913 directly
under the thermal interface laminate, shown in cross-section. In one
embodiment the heaters are photolithographically defined and etched metal
layers of gold (typically about 3,000 Å thick). Layers of 400 Å
of TiW (not shown) are deposited on top and bottom of the gold layer to
serve as an adhesion layer. The substrate 913 used is glass, fused silica
or a quartz wafer having a thickness of 0.4 mm, 0.5 mm, 0.7 mm, or 1 mm.
A thin electrically-insulative layer of 2 μm silicon oxide serves as
an insulative layer on top of the metal layer. Additional thin
electrically insulative layers such as 2-4 μm of Parylene may also be
deposited on top of the silicon oxide surface. Two long heaters 909 and
911, as further described herein, run alongside the PCR channel.

[0116]An exemplary heater array is shown in FIGS. 15A and 15B. Additional
embodiments of heater arrays are described in U.S. patent application
Ser. No. ______, entitled "Heater Unit for Microfluidic Diagnostic
System" and filed on even date herewith, the specification of which is
incorporated herein by reference in its entirety.

[0117]Referring to FIGS. 15A and 15B, an exemplary PCR reaction chamber
1501, typically having a volume ˜1.6 μl, is configured with a
long side and a short side, each with an associated heating element. The
heater substrate therefore includes four heaters disposed along the sides
of, and configured to heat, the PCR reaction chamber, as shown in the
exemplary embodiment of FIG. 15A: long top heater 1505, long bottom
heater 1503, short left heater 1507, and short right heater 1509. The
small gap between long top heater 1505 and long bottom heater 1503
results in a negligible temperature gradient (less than 1° C.
difference across the width of the PCR channel at any point along the
length of the PCR reaction chamber) and therefore an effectively uniform
temperature throughout the PCR reaction chamber. The heaters on the short
edges of the PCR reactor provide heat to counteract the gradient created
by the two long heaters from the center of the reactor to the edge of the
reactor. It would be understood by one of ordinary skill in the art that
still other configurations of one or more heater(s) situated about a PCR
reaction chamber are consistent with the methods and apparatus described
herein. For example, a `long` side of the reaction chamber can be
configured to be heated by two or more heaters. Specific orientations and
configurations of heaters are used to create uniform zones of heating
even on substrates having poor thermal conductivity because the poor
thermal conductivity of glass, or quartz, polyimide, FR4, ceramic, or
fused silica substrates is utilized to help in the independent operation
of various microfluidic components such as valves and independent
operation of the various PCR lanes. In FIG. 15B, various aspects of fine
structure of heater elements are shown in inserts.

[0118]Generally, the heating of microfluidic components, such as a PCR
reaction chamber, is controlled by passing currents through suitably
configured microfabricated heaters. Under control of suitable circuitry,
the lanes of a multi-lane cartridge can then be controlled independently
of one another. This can lead to a greater energy efficiency of the
apparatus, because not all heaters are heating at the same time, and a
given heater is receiving current for only that fraction of the time when
it is required to heat. Control systems and methods of controllably
heating various heating elements are further described in U.S. patent
application Ser. No. ______, filed Nov. 14, 2007 and entitled "Heater
Unit for Microfluidic Diagnostic System".

[0119]The configuration for uniform heating, shown in FIG. 15A for a
single PCR reaction chamber, can be applied to a multi-lane PCR cartridge
in which multiple independent PCR reactions occur. See, e.g., FIG. 15c,
which shows an array of heater elements suitable to heat the cartridge of
FIG. 1.

[0120]In other embodiments, as further described in U.S. patent
application Ser. No. ______, filed Nov. 14, 2007 and entitled "Heater
Unit for Microfluidic Diagnostic System", the heaters may have an
associated temperature sensor, or may themselves function as sensors.

Use of Cutaways in Cartridge and Substrate to Improve Rate of Cooling
During PCR Cycling

[0121]During a PCR amplification of a nucleotide sample, a number of
thermal cycles are carried out. For improved efficiency, the cooling
between each application of heat is preferably as rapid as possible.
Improved rate of cooling can be achieved with various modifications to
the heating substrate and/or the cartridge, as shown in FIG. 16.

[0122]One way to achieve rapid cooling is to cutaway portions of the
microfluidic cartridge substrate, as shown in FIG. 16. The upper panel of
FIG. 16 is a cross-section of an exemplary microfluidic cartridge taken
along the dashed line A-A' as marked on the lower panel of FIG. 16. PCR
reaction chamber 1601, and representative heaters 1603 are shown. Also
shown are two cutaway portions, one of which labeled 1601, that are
situated alongside the heaters that are positioned along the long side of
the PCR reaction chamber. Cutaway portions such as 1601 reduce the
thermal mass of the cartridge, and also permit air to circulate within
the cutaway portions. Both of these aspects permit heat to be conducted
away quickly from the immediate vicinity of the PCR reaction chamber.
Other configurations of cutouts, such as in shape, position, and number,
are consistent with the present technology.

[0123]Another way to achieve rapid cooling is to cutaway portions of the
heater substrate, and also to use ambient air cooling, as further
described in U.S. patent application Ser. No. ______, filed Nov. 14, 2007
and entitled "Heater Unit for Microfluidic Diagnostic System".

[0124]An example of thermal cycling performance in a PCR reaction chamber
obtained with a configuration as described herein, is shown in FIG. 17
for a protocol that is set to heat up the reaction mixture to 92°
C., and maintain the temperature for 1 second, then cool to 62°
C., and stay for 10 seconds. The cycle time shown is about 29 seconds,
with 8 seconds required to heat from 62° C. and stabilize at
92° C., and 10 seconds required to cool from 92° C., and
stabilize at 62° C. To minimize the overall time required for a
PCR effective to produce detectable quantities of amplified material, it
is important to minimize the time required for each cycle. Cycle times in
the range 15-30 s, such as 18-25 s, and 20-22 s, are desirable. In
general, an average PCR cycle time of 25 seconds as well as cycle times
as low as 20 seconds are typical with the technology described herein.
Using reaction volumes less than a microliter (such as a few hundred
nanoliters or less) permits use of an associated smaller PCR chamber, and
enables cycle times as low as 15 seconds.

Manufacturing Process for Cartridge

[0125]FIG. 18 shows a flow-chart 1800 for an embodiment of an assembly
process for an exemplary cartridge as shown in FIG. 4A herein. It would
be understood by one of ordinary skill in the art, both that various
steps may be performed in a different order from the order set forth in
FIG. 18, and additionally that any given step may be carried out by
alternative methods to those described in the figure. It would also be
understood that, where separate serial steps are illustrated for carrying
out two or more functions, such functions may be performed synchronously
and combined into single steps and remain consistent with the overall
process described herein.

[0126]At 1802, a laminate layer is applied to a microfluidic substrate
that has previously been engineered, for example by injection molding, to
have a microfluidic network constructed in it; edges are trimmed from the
laminate where they spill over the bounds of the substrate.

[0127]At 1804, wax is dispensed and loaded into the microvalves of the
microfluidic network in the microfluidic substrate. An exemplary process
for carrying this out is further described herein.

[0128]At 1806, the substrate is inspected to ensure that wax from step
1804 is loaded properly and that the laminate from step 1802 adheres
properly to it. If a substrate does not satisfy either or both of these
tests, it is usually discarded. If substrates repeatedly fail either or
both of these tests, then the wax dispensing, or laminate application
steps, as applicable, are reviewed.

[0129]At 1808, a hydrophobic vent membrane is applied to, and heat bonded
to, the top of the microfluidic substrate covering at least the one or
more vent holes, and on the opposite face of the substrate from the
laminate. Edges of the membrane that are in excess of the boundary of the
substrate are trimmed.

[0130]At 1810, the assembly is inspected to ensure that the hydrophobic
vent membrane is bonded well to the microfluidic substrate without
heat-clogging the microfluidic channels. If any of the channels is
blocked, or if the bond between the membrane and the substrate is
imperfect, the assembly is discarded, and, in the case of repeated
discard events, the foregoing process step 1808 is reviewed.

[0131]At 1812, optionally, a thermally conductive pad layer is applied to
the bottom laminate of the cartridge.

[0132]At 1814, two label strips are applied to the top of the microfluidic
substrate, one to cover the valves, and a second to protect the vent
membranes. It would be understood that a single label strip may be
devised to fulfill both of these roles.

[0133]At 1816, additional labels are printed or applied to show
identifying characteristics, such as a barcode #, lot # and expiry date
on the cartridge. Preferably one or more of these labels has a space and
a writable surface that permits a user to make an identifying annotation
on the label, by hand.

[0134]Optionally, at 1818, to facilitate transport and delivery to a
customer, assembled and labeled cartridges are stacked, and cartridges
packed into groups, such as groups of 25, or groups of 10, or groups of
20, or groups of 48 or 50. Preferably the packaging is via an inert
and/or moisture-free medium.

Wax Loading in Valves

[0135]In general, a valve as shown in, e.g., FIGS. 12A-C, is constructed
by depositing a precisely controlled amount of a TRS (such as wax) into a
loading inlet machined in the microfluidic substrate. FIGS. 19A and 19B
show how a combination of controlled hot drop dispensing into a heated
microchannel device of the right dimensions and geometry is used to
accurately load wax into a microchannel of a microfluidic cartridge to
form a valve. The top of FIG. 19A shows a plan view of a valve inlet 190
and loading channel 1902, connecting to a flow channel 1904. The lower
portions of FIG. 19A show the progression of a dispensed wax droplet 1906
(having a volume of 75 nl±15 nl) through the inlet 1901 and into the
loading channel 1902.

[0136]To accomplish those steps, a heated dispenser head can be accurately
positioned over the inlet hole of the microchannel in the microfluidic
device, and can dispense molten wax drops in volumes as small as 75
nanoliters with an accuracy of 20%. A suitable dispenser is also one that
can deposit amounts smaller than 100 nl with a precision of +/-20%. The
dispenser should also be capable of heating and maintaining the
dispensing temperature of the TRS to be dispensed. For example, it may
have a reservoir to hold the solution of TRS. It is also desirable that
the dispense head can have freedom of movement at least in a horizontal
(x-y) plane so that it can easily move to various locations of a
microfluidic substrate and dispense volumes of TRS into valve inlets at
such locations without having to be re-set, repositioned manually, or
recalibrated in between each dispense operation.

[0137]The inlet hole of the microfluidic cartridge, or other microchannel
device, is dimensioned in such a way that the droplet of 75 nl can be
accurately propelled to the bottom of the inlet hole using, for example,
compressed air, or in a manner similar to an inkjet printing method. The
microfluidic cartridge is maintained at a temperature above the melting
point of the wax thereby permitting the wax to stay in a molten state
immediately after it is dispensed. After the drop falls to the bottom of
the inlet hole 1901, the molten wax is drawn into the narrow channel by
capillary action, as shown in the sequence of views in FIG. 19B. A
shoulder between the inlet hole 1901 and the loading channel can
facilitate motion of the TRS. The volume of the narrow section can be
designed to be approximately equal to a maximum typical amount that is
dispensed into the inlet hole. The narrow section can also be designed so
that even though the wax dispensed may vary considerably between a
minimum and a maximum shot size, the wax always fills up to, and stops
at, the microchannel junction 1907 because the T-junction provides a
higher cross section than that of the narrow section and thus reduces the
capillary forces. Dimensions shown in FIG. 19A are exemplary.

PCR Reagent Mixtures

[0138]In various embodiments, the sample for introduction into a lane of
the microfluidic cartridge can include a PCR reagent mixture comprising a
polymerase enzyme and a plurality of nucleotides.

[0139]In various embodiments, preparation of a PCR-ready sample for use
with an apparatus and cartridge as described herein can include
contacting a neutralized polynucleotide sample with a PCR reagent mixture
comprising a polymerase enzyme and a plurality of nucleotides (in some
embodiments, the PCR reagent mixture can further include a positive
control plasmid and a fluorogenic hybridization probe selective for at
least a portion of the plasmid).

[0141]The PCR reagent mixture can be in the form of one or more
lyophilized pellets and the steps by which the PCR-ready sample is
prepared can involve reconstituting the PCR pellet by contacting it with
liquid to create a PCR reagent mixture solution. In yet another
embodiment, each of the PCR lanes may have dried down or lyophilized ASR
reagents preloaded such that the user only needs to input prepared
polynucleotide sample into the PCR. In another embodiment, the PCR lanes
may have only the application-specific probes and primers pre-measured
and pre-loaded, and the user inputs a sample mixed with the PCR reagents.

[0142]In various embodiments, the PCR-ready sample can include at least
one probe that can be selective for a polynucleotide sequence, wherein
the steps by which the PCR-ready sample is prepared involve contacting
the neutralized polynucleotide sample or a PCR amplicon thereof with the
probe. The probe can be a fluorogenic hybridization probe. The
fluorogenic hybridization probe can include a polynucleotide sequence
coupled to a fluorescent reporter dye and a fluorescence quencher dye.

[0143]In various embodiments, the PCR-ready sample further includes a
sample buffer.

[0144]In various embodiments, the PCR-ready sample includes at least one
probe that is selective for a polynucleotide sequence, e.g., the
polynucleotide sequence that is characteristic of a pathogen selected
from the group consisting of gram positive bacteria, gram negative
bacteria, yeast, fungi, protozoa, and viruses.

[0145]In various embodiments, the PCR reagent mixture can further include
a polymerase enzyme, a positive control plasmid and a fluorogenic
hybridization probe selective for at least a portion of the plasmid.

[0146]In various embodiments, the probe can be selective for a
polynucleotide sequence that is characteristic of an organism, for
example any organism that employs deoxyribonucleic acid or ribonucleic
acid polynucleotides. Thus, the probe can be selective for any organism.
Suitable organisms include mammals (including humans), birds, reptiles,
amphibians, fish, domesticated animals, wild animals, extinct organisms,
bacteria, fungi, viruses, plants, and the like. The probe can also be
selective for components of organisms that employ their own
polynucleotides, for example mitochondria. In some embodiments, the probe
is selective for microorganisms, for example, organisms used in food
production (for example, yeasts employed in fermented products, molds or
bacteria employed in cheeses, and the like) or pathogens (e.g., of
humans, domesticated or wild mammals, domesticated or wild birds, and the
like). In some embodiments, the probe is selective for organisms selected
from the group consisting of gram positive bacteria, gram negative
bacteria, yeast, fungi, protozoa, and viruses.

[0149]In various embodiments, the probe can be selective for a
polynucleotide sequence that is characteristic of Group B Streptococcus.

[0150]In various embodiments, a method of carrying out PCR on a sample can
further include one of more of the following steps: heating the
biological sample in the microfluidic cartridge; pressurizing the
biological sample in the microfluidic cartridge at a pressure
differential compared to ambient pressure of between about 20 kilopascals
and 200 kilopascals, or in some embodiments, between about 70 kilopascals
and 110 kilopascals.

[0151]In some embodiments, the method for sampling a polynucleotide can
include the steps of: placing a microfluidic cartridge containing a
PCR-ready sample in a receiving bay of a suitably configured apparatus;
carrying out PCR on the sample under thermal cycling conditions suitable
for creating PCR amplicons from the neutralized polynucleotide in the
sample, the PCR-ready sample comprising a polymerase enzyme, a positive
control plasmid, a fluorogenic hybridization probe selective for at least
a portion of the plasmid, and a plurality of nucleotides; contacting the
neutralized polynucleotide sample or a PCR amplicon thereof with the at
least one fluorogenic probe that is selective for a polynucleotide
sequence, wherein the probe is selective for a polynucleotide sequence
that is characteristic of an organism selected from the group consisting
of gram positive bacteria, gram negative bacteria, yeast, fungi,
protozoa, and viruses; and detecting the fluorogenic probe, the presence
of the organism for which the one fluorogenic probe is selective is
determined.

[0152]Carrying out PCR on a PCR-ready sample can additionally include:
independently contacting each of the neutralized polynucleotide sample
and a negative control polynucleotide with the PCR reagent mixture under
thermal cycling conditions suitable for independently creating PCR
amplicons of the neutralized polynucleotide sample and PCR amplicons of
the negative control polynucleotide; and/or contacting the neutralized
polynucleotide sample or a PCR amplicon thereof and the negative control
polynucleotide or a PCR amplicon thereof with at least one probe that is
selective for a polynucleotide sequence.

[0153]In various embodiments, a method of using the apparatus and
cartridge described herein can further include one or more of the
following steps: determining the presence of a polynucleotide sequence in
the biological sample, the polynucleotide sequence corresponding to the
probe, if the probe is detected in the neutralized polynucleotide sample
or a PCR amplicon thereof; determining that the sample was contaminated
if the probe is detected in the negative control polynucleotide or a PCR
amplicon thereof; and/or in some embodiments, wherein the PCR reagent
mixture further comprises a positive control plasmid and a plasmid probe
selective for at least a portion of the plasmid, the method further
including determining that a PCR amplification has occurred if the
plasmid probe is detected.

Kit

[0154]In various embodiments, the microfluidic cartridge as described
herein can be provided in the form of a kit, wherein the kit can include
a microfluidic cartridge, and a liquid transfer member (such as a syringe
or a pipette). In various embodiments, the kit can further include
instructions to employ the liquid transfer member to transfer a sample
containing extracted nucleic acid from a sample container via a sample
inlet to the microfluidic network on the microfluidic cartridge. In some
embodiments, the microfluidic cartridge and the liquid transfer member
can be sealed in a pouch with an inert gas.

[0155]Typically when transferring a sample from liquid dispenser, such as
a pipette tip, to an inlet on the microfluidic cartridge, a volume of air
is simultaneously introduced into the microfluidic network, the volume of
air being between about 0.5 mL and about 5 mL. Presence of air in the
microfluidic network, however, is not essential to operation of the
cartridge described herein.

[0156]In various embodiments, the kit can further include at least one
computer-readable label on the cartridge. The label can include, for
example, a bar code, a radio frequency tag or one or more
computer-readable characters. When used in conjunction with a similar
computer-readable label on a sample container, such as a vial or a pouch,
matching of diagnostic results with sample is thereby facilitated.

[0157]In some embodiments, a sample identifier of the apparatus described
elsewhere herein is employed to read a label on the microfluidic
cartridge and/or a label on the biological sample.

Overview of an Apparatus for Receiving a Microfluidic Cartridge

[0158]The present technology relates to a cartridge, complementary
apparatus, and related methods for amplifying, and carrying out
diagnostic analyses on, nucleotides from biological samples. The
technology includes a disposable or reusable microfluidic cartridge
containing multiple sample lanes capable of processing samples in
parallel as described elsewhere herein, and a reusable apparatus that is
configured to selectively actuate on-cartridge operations, to detect and
analyze the products of the PCR amplification in each of the lanes
separately, in all simultaneously, or in groups simultaneously, and,
optionally, can display the progression of analyses and results thereof
on a graphical user interface. Such a reusable apparatus is further
described in U.S. patent application Ser. No. ______, entitled
"Microfluidic System for Amplifying and Detecting Polynucleotides in
Parallel" and filed on Nov. 14, 2007, and which is incorporated herein by
reference in its entirety.

[0159]FIG. 20 shows a perspective view of an exemplary apparatus 2000
consistent with those described herein, as well as various components
thereof, such as exemplary cartridge 2010 that contains multiple sample
lanes, and exemplary read head 2020 that contains detection apparatus for
reading signals from cartridge 2010. The apparatus 2000 of FIG. 20 is
able to carry out real-time PCR on a number of samples in cartridge 2010
simultaneously or serially. Preferably the number of samples is 12
samples, as illustrated with exemplary cartridge 2010, though other
numbers of samples such as 4, 8, 10, 16, 20, 24, 25, 30, 32, 36, 40, and
48 are within the scope of the present description. In preferred
operation of the apparatus, a PCR-ready solution containing the sample,
and, optionally, one or more analyte-specific reagents (ASR's) is
prepared, as further described elsewhere (see, e.g., U.S. patent
application publication 2006-0166233, incorporated herein by reference),
prior to introduction into cartridge 200.

[0160]In some embodiments, an apparatus includes: a receiving bay
configured to selectively receive a microfluidic cartridge as described
herein; at least one heat source thermally coupled to the receiving bay;
and a processor coupled to the heat source, wherein the heat source is
configured to selectively heat individual regions of individual sample
lanes in the cartridge, and the processor is configured to control
application of heat to the individual sample lanes, separately, in all
simultaneously, or in groups simultaneously; at least one detector
configured to detect one or more polynucleotides or a probe thereof in a
sample in one or more of the individual sample lanes, separately or
simultaneously; and a processor coupled to the detector to control the
detector and to receive signals from the detector.

[0161]FIG. 21 shows a schematic cross-sectional view of a part of an
apparatus as described herein, showing input of sample into a cartridge
2100 via a pipette 10 (such as a disposable pipette) and an inlet 202.
Cartridge 2100 is situated in a suitably configured receiving bay 2112.
Inlet 2102 is preferably configured to receive a pipette or the bottom
end of a PCR tube and thereby accept sample for analysis with minimum
waste, and with minimum introduction of air. Cartridge 2100 is disposed
on top of and in contact with a heater substrate 2140. Read head 2130 is
positioned above cartridge 2100 and a cover for optics 2131 restricts the
amount of ambient light that can be detected by the read head.

[0162]FIG. 22 shows an example of 4-pipette head used for attaching
disposable pipette tips, prior to dispensing PCR-ready sample into a
cartridge as further described herein.

[0163]The receiving bay is a portion of the apparatus that is configured
to selectively receive the microfluidic cartridge. For example, the
receiving bay and the microfluidic cartridge can be complementary in
shape so that the microfluidic cartridge is selectively received in,
e.g., a single orientation. The microfluidic cartridge can have a
registration member that fits into a complementary feature of the
receiving bay. The registration member can be, for example, a cut-out on
an edge of the cartridge, such as a corner that is cut-off, or one or
more notches or grooves that are made on one or more of the sides in a
distinctive pattern that prevents a cartridge from being loaded into the
bay in more than one distinct orientation. By selectively receiving the
cartridge, the receiving bay can help a user to place the cartridge so
that the apparatus can properly operate on the cartridge. The cartridge
can be designed to be slightly smaller than the dimensions of the
receiving bay, for example by approximately 200-300 microns, for easy
placement and removal of the cartridge.

[0164]The receiving bay can also be configured so that various components
of the apparatus that operate on the microfluidic cartridge (heat
sources, detectors, force members, and the like) are positioned to
properly operate thereon. For example, a contact heat source can be
positioned in the receiving bay such that it can be thermally coupled to
one or more distinct locations on a microfluidic cartridge that is
selectively received in the bay. Microheaters in the heater module as
further described elsewhere herein were aligned with corresponding
heat-requiring microcomponents (such as valves, pumps, gates, reaction
chambers, etc). The microheaters can be designed to be slightly bigger
than the heat requiring microfluidic components so that even though the
cartridge may be off-centered from the heater, the individual components
can still function effectively.

[0165]As further described elsewhere herein, the lower surface of the
cartridge can have a layer of mechanically compliant heat transfer
laminate that can enable thermal contact between the microfluidic
substrate and the microheater substrate of the heater module. A minimal
pressure of 1 psi can be employed for reliable operation of the thermal
valves, gates and pumps present in the microfluidic cartridge.

[0166]In various embodiments of the apparatus, the apparatus can further
include a sensor coupled to the processor, the sensor configured to sense
whether the microfluidic cartridge is selectively received.

[0167]The heat source can be, for example, a heat source such as a
resistive heater or network of resistive heaters. In preferred
embodiments, the at least one heat source can be a contact heat source
selected from a resistive heater (or network thereof), a radiator, a
fluidic heat exchanger and a Peltier device. The contact heat source can
be configured at the receiving bay to be thermally coupled to one or more
distinct locations of a microfluidic cartridge received in the receiving
bay, whereby the distinct locations are selectively heated. The contact
heat source typically includes a plurality of contact heat sources, each
configured at the receiving bay to be independently thermally coupled to
a different distinct location in a microfluidic cartridge received
therein, whereby the distinct locations are independently heated. The
contact heat sources can be configured to be in direct physical contact
with one or more distinct locations of a microfluidic cartridge received
in the bay. In various embodiments, each contact source heater can be
configured to heat a distinct location having an average diameter in 2
dimensions from about 1 millimeter (mm) to about 15 mm (typically about 1
mm to about 10 mm), or a distinct location having a surface area of
between about 1 mm2 about 225 mm (typically between about 1 mm2
and about 100 mm2, or in some embodiments between about 5 mm and
about 50 mm2). Various configurations of heat sources are further
described in U.S. patent application Ser. No. ______, entitled "Heater
Unit for Microfluidic Diagnostic System" and filed on even date herewith.

[0168]In various embodiments, the heat source is disposed in a heating
module that is configured to be removable from the apparatus.

[0169]In various embodiments, the apparatus can include a compliant layer
at the contact heat source configured to thermally couple the contact
heat source with at least a portion of a microfluidic cartridge received
in the receiving bay. The compliant layer can have a thickness of between
about 0.05 and about 2 millimeters and a Shore hardness of between about
25 and about 100. Such a compliant layer may not be required if the
instrument is able to reliably press the cartridge over the heater
surface with a minimum contact pressure of say 1 psi over the entirety of
the cartridge.

[0170]The detector can be, for example, an optical detector. For example,
the detector can include a light source that selectively emits light in
an absorption band of a fluorescent dye, and a light detector that
selectively detects light in an emission band of the fluorescent dye,
wherein the fluorescent dye corresponds to a fluorescent polynucleotide
probe or a fragment thereof. Alternatively, for example, the optical
detector can include a bandpass-filtered diode that selectively emits
light in the absorption band of the fluorescent dye and a bandpass
filtered photodiode that selectively detects light in the emission band
of the fluorescent dye; or for example, the optical detector can be
configured to independently detect a plurality of fluorescent dyes having
different fluorescent emission spectra, wherein each fluorescent dye
corresponds to a fluorescent polynucleotide probe or a fragment thereof;
or for example, the optical detector can be configured to independently
detect a plurality of fluorescent dyes at a plurality of different
locations on a microfluidic cartridge, wherein each fluorescent dye
corresponds to a fluorescent polynucleotide probe or a fragment thereof
in a different sample. The detector can also be configured to detect the
presence or absence of sample in a PCR reaction chamber in a given sample
lane, and to condition initiation of thermocycling upon affirmative
detection of presence of the sample. Further description of suitably
configured detectors are described in U.S. patent application Ser. No.
______, filed on Nov. 14, 2007 and entitled "Fluorescence Detector for
Microfluidic Diagnostic System", incorporated herein by reference.

[0171]Although the various depictions therein show a heater substrate
disposed underneath a microfluidic substrate, and a detector disposed on
top of it, it would be understood that an inverted arrangement would work
equally as well. In such an embodiment, the heater would be forced down
onto the microfluidic substrate, making contact therewith, and the
detector would be mounted underneath the substrate, disposed to collect
light directed downwards towards it.

[0172]In another preferred embodiment (not shown in the FIGs. herein), a
cartridge and apparatus are configured so that the read-head does not
cover the sample inlets, thereby permitting loading of separate samples
while other samples are undergoing PCR thermocycling.

[0173]In various embodiments, the apparatus can further include an
analysis port. The analysis port can be configured to allow an external
sample analyzer to analyze a sample in the microfluidic cartridge. For
example, the analysis port can be a hole or window in the apparatus which
can accept an optical detection probe that can analyze a sample or
progress of PCR in situ in the microfluidic cartridge. In some
embodiments, the analysis port can be configured to direct a sample from
the microfluidic cartridge to an external sample analyzer; for example,
the analysis port can include a conduit in fluid communication with the
microfluidic cartridge that directs a liquid sample containing an
amplified polynucleotide to a chromatography apparatus, an optical
spectrometer, a mass spectrometer, or the like.

[0174]In various embodiments, the apparatus can further include one or
more force members configured to apply force to at least a portion of a
microfluidic cartridge received in the receiving bay. The one or more
force members are configured to apply force to thermally couple the at
least one heat source to at least a portion of the microfluidic
cartridge. The application of force is important to ensure consistent
thermal contact between the heater wafer and the PCR reactor and
microvalves in the microfluidic cartridge.

[0175]The apparatus preferably also includes a processor, comprising
microprocessor circuitry, in communication with, for example, the input
device and a display, that accepts a user's instructions and controls
analysis of samples.

[0176]In various embodiments, the apparatus can further include a lid at
the receiving bay, the lid being operable to at least partially exclude
ambient light from the receiving bay.

[0177]In various embodiments, the apparatus can further include at least
one input device coupled to the processor, the input device being
selected from the group consisting of a keyboard, a touch-sensitive
surface, a microphone, and a mouse.

[0178]In various embodiments, the apparatus can further include at least
one sample identifier coupled to the processor, the sample identifier
being selected from an optical scanner such as an optical character
reader, a bar code reader, or a radio frequency tag reader. For example,
the sample identifier can be a handheld bar code reader.

[0179]In various embodiments, the apparatus can further include at least
one data storage medium coupled to the processor, the medium selected
from: a hard disk drive, an optical disk drive, or one or more removable
storage media such as a CD-R, CD-RW, USB-drive, or flash memory card.

[0180]In various embodiments, the apparatus can further include at least
one output coupled to the processor, the output being selected from a
display, a printer, and a speaker, the coupling being either directly
through a directly dedicated printer cable, or wirelessly, or via a
network connection.

[0181]The apparatus further optionally comprises a display that
communicates information to a user of the system. Such information
includes but is not limited to: the current status of the system;
progress of PCR thermocycling; and a warning message in case of
malfunction of either system or cartridge. The display is preferably used
in conjunction with an external input device as elsewhere described
herein, through which a user may communicate instructions to apparatus
100. A suitable input device may further comprise a reader of formatted
electronic media, such as, but not limited to, a flash memory card,
memory stick, USB-stick, CD, or floppy diskette. An input device may
further comprise a security feature such as a fingerprint reader, retinal
scanner, magnetic strip reader, or bar-code reader, for ensuring that a
user of the system is in fact authorized to do so, according to
pre-loaded identifying characteristics of authorized users. An input
device may additionally--and simultaneously--function as an output device
for writing data in connection with sample analysis. For example, if an
input device is a reader of formatted electronic media, it may also be a
writer of such media. Data that may be written to such media by such a
device includes, but is not limited to, environmental information, such
as temperature or humidity, pertaining to an analysis, as well as a
diagnostic result, and identifying data for the sample in question.

[0182]The apparatus may further include a computer network connection that
permits extraction of data to a remote location, such as a personal
computer, personal digital assistant, or network storage device such as
computer server or disk farm. The network connection can be a
communications interface selected from the group consisting of: a serial
connection, a parallel connection, a wireless network connection, and a
wired network connection such as an ethernet or cable connection, wherein
the communications interface is in communication with at least the
processor. The computer network connection may utilize, e.g., ethernet,
firewire, or USB connectivity. The apparatus may further be configured to
permit a user to e-mail results of an analysis directly to some other
party, such as a healthcare provider, or a diagnostic facility, or a
patient.

[0183]In various embodiments, there is an associated computer program
product includes computer readable instructions thereon for operating the
apparatus and for accepting instructions from a user.

[0184]In various embodiments, the computer program product can include one
or more instructions to cause the system to: output an indicator of the
placement of the microfluidic cartridge in the receiving bay; read a
sample label or a microfluidic cartridge label; output directions for a
user to input a sample identifier; output directions for a user to load a
sample transfer member with the PCR-ready sample; output directions for a
user to introduce the PCR-ready sample into the microfluidic cartridge;
output directions for a user to place the microfluidic cartridge in the
receiving bay; output directions for a user to close the lid to operate
the force member; output directions for a user to pressurize the
PCR-ready sample in the microfluidic cartridge by injecting the PCR-ready
sample with a volume of air between about 0.5 mL and about 5 mL; and
output status information for sample progress from one or more lanes of
the cartridge.

[0185]In various embodiments, the computer program product can include one
or more instructions to cause the system to: heat the PCR ready-sample
under thermal cycling conditions suitable for creating PCR amplicons from
the neutralized polynucleotide; contact the neutralized polynucleotide
sample or a PCR amplicon thereof with at least one probe that is
selective for a polynucleotide sequence; independently contact each of
the neutralized polynucleotide sample and a negative control
polynucleotide with the PCR reagent mixture under thermal cycling
conditions suitable for independently creating PCR amplicons of the
neutralized polynucleotide sample and PCR amplicons of the negative
control polynucleotide; contact the neutralized polynucleotide sample or
a PCR amplicon thereof and the negative control polynucleotide or a PCR
amplicon thereof with at least one probe that is selective for a
polynucleotide sequence; output a determination of the presence of a
polynucleotide sequence in the biological sample, the polynucleotide
sequence corresponding to the probe, if the probe is detected in the
neutralized polynucleotide sample or a PCR amplicon thereof; and/or
output a determination of a contaminated result if the probe is detected
in the negative control polynucleotide or a PCR amplicon thereof.

[0186]Apparatus 100 may optionally comprise one or more stabilizing feet
that cause the body of the device to be elevated above a surface on which
system 100 is disposed, thereby permitting ventilation underneath system
100, and also providing a user with an improved ability to lift system
100.

EXAMPLES

Example 1

48 Lane Cartridge

[0187]FIG. 23 shows an exemplary 48-lane cartridge for carrying out PCR
independently on 48 samples, and with a reaction volume of 10 microliter
each. The area occupied by the entire cartridge is approximately 3.5
inches (8.9 cm) by 4.25 inches (10.8 cm). The sample lanes are organized
as two groups of 24 each. The adjacent sample lanes in each of the two
rows of 24 are spaced apart 4 mm (center-to-center). Trenches between the
PCR lanes may be cut in order to isolate the heating of each PCR channel
from those adjacent to it. This may be accomplished by etching, milling,
controlled cutting, etc., during fabrication of the cartridge.

[0188]FIG. 24 shows a heater design used for actuating the 48 lane PCR
cartridge of FIG. 23. The heating of each sample lane can be
independently controlled.

Example 2

PCR Cartridge with Post-PCR Retrieval Capability

[0189]Many applications such as genotyping, sequencing, multiple analyte
detection (microarray, electrochemical sensing) require post-PCR sample
retrieval and subsequent analysis of the retrieved sample in a different
instrument. The cartridge of this example, of which a 24 lane embodiment
is shown in FIG. 25A, with a sample lane layout illustrated in FIG. 25B,
accommodates such a retrieval capability. Each lane in the cartridge of
FIG. 25A independently permits sample retrieval. The configuration of the
lane of FIG. 25B is different from that of, e.g., FIG. 6 at least because
of the presence of 2 gates and the alternative channel from the reactor,
via Gate 1, to the inlet. Such features permit effective sample
retrieval.

[0190]Sample DNA mixed with PCR enzymes is input into a sample lane 2501
through the inlet hole 2502 in the microfluidic network described below.
The valves 2506, 2504 (valves 1 and 2) are initially open while the gates
2522, 2520 (gates 1 and 2) are closed, enabling the reaction mix to fill
up the PCR reactor 2510 with the excess air venting out through vent hole
1 (label 2514). The valves 1 and 2 are then closed to seal off the
reaction mixture. Thermocycling is initiated to conduct the PCR reaction
within the PCR reactor. After the reaction is completed, a pipette is
mechanically interfaced with the inlet hole 2502 and suction force
applied to the pipette. Gates 1 and 2 are opened to enable the reacted
sample to exit the PCR reactor and enter the pipette. This controlled
opening of the PCR device will also prevent post-PCR contamination of the
apparatus in which the cartridge resides as there is minimal exposure of
the PCR product with the atmosphere.

[0191]It will be understood that reactions other than PCR can easily be
performed in the cartridge of this example.

Example 3

12-Lane Cartridge

[0192]The 12 channel cartridge of this example is the same basic design
that is described and shown in FIG. 3, with the following modifications:
the volume of the PCR reactor is increased from 2 μl to 4.5 μl,
leading to an increase in the acceptable input volume from 4 μl to 6
μl. Increasing the reaction volume facilitates detection from even
dilute samples (wherein the target DNA concentration may be low). In
order to detect DNA in a reactor of say 1 microliter volume, there should
be a minimum of 1-2 copies of the DNA in the 1 microliter for positive
identification, i.e., the concentration should not be less than around
1-2 copies/microliter. Increasing the reaction volume to say 5
microliters will reduce the minimum acceptable starting DNA concentration
by 5 fold. The inlet holes are moved a few millimeters away from the edge
of the cartridge to allow room for a 2 mm alignment ledge in the
cartridge. A similar alignment ledge is also included on the other edge
of the cartridge. The alignment ledge permits the cartridges to be
stacked during storage (or within a multi-cartridge spring-loader)
without the hydrophobic vent of one cartridge coming into contact with a
surface of an adjacent cartridge.

Example 4

24-Lane Cartridge

[0193]This 24-lane cartridge has two rows of 12 sample lanes. Each lane
has: a liquid inlet port, that interfaces with a disposable pipette; a 4
microliter PCR reaction chamber (1.5 mm wide, 300 microns deep and
approximately 10 mm long), and two microvalves on either side of the PCR
reactor and outlet vent. Microvalves are normally open, and close the
channel on actuation. The outlet holes enable extra liquid (˜1
μl) to be contained in the fluidic channel in case more than 6 μl
of fluid is dispensed into the cartridge. Thus, the cartridge of this
example does not require a bubble vent as it will be used in an automated
PCR machine having a reliable, precision liquid dispenser.

[0194]The inlet holes of the cartridge of this example are made conical in
shape and have a diameter of 3-6 mm at the top to ensure that pipette
tips can be easily landed by an automated fluid dispensing head into the
conical hole, with some tolerance. There is also an optional raised
annulus around the top of the holes. Once the pipette tip lands within
the cone, the conical shape guides the pipette and mechanically seals to
provide error free dispensing into, or withdrawal of fluid from, the
cartridge. The bigger the holes, the better it is to align with the
pipette, however, given the opposing need to maximize the number of inlet
ports within the width of the cartridge as well as to maintain the pitch
between holes compatible with the inter-pipette distance, the holes
cannot be too big. In this design, the inter-pipette tip distance is 18
mm and the distance between the loading holes in the cartridge is 6 mm.
So lanes 1, 4, 7, 11 are pipetted into during one dispensing operation
that utilizes four pipette tips; lanes 2, 5, 8 and 12 in the next, and so
on and so forth.

[0195]The height of the conical holes is kept lower than the height of the
ledges on the edges of the cartridge to ensure the cartridges can be
stacked on the ledges. The ledges on the two long edges of the cartridge
enable stacking of the cartridges with minimal surface contact between
two stacked cartridges and also help guide the cartridge into the reader
from a spring-loader, where used.

Example 5

12-Lane Cartridge

[0196]This 12-lane cartridge has 12 sample lanes in parallel, as shown in
FIG. 1. Each lane has: a liquid inlet port that interfaces with a
disposable pipette; a bubble vent; a PCR reaction chamber, and two
microvalves on either side of the PCR reactor and outlet vent.
Microvalves are normally open, and close the channel on actuation. The
reaction volume is in the range 1-10 μl so that the number of copies
of DNA will be sufficient for detection. Such a volume also permits the
PCR reaction volume to be similar to release volume from a sample
preparation procedure.

Example 6

Kit

[0197]FIG. 26 shows a representative sample kit 2610 that includes a
microfluidic cartridge 2612 with a barcode label 2632, and one or more
sample containers 2614 each also optionally having a barcode label.

[0198]FIG. 27 shows that one or more components of the sample kit, for
example, microfluidic cartridge 2612, can be packaged in a sealed pouch
2624. The pouch can be hermetically sealed with an inert gas such as
argon, nitrogen, or others.

[0199]The barcode labels of both cartridge and sample container can be
read with a bar code reader prior to use.

Example 7

Apparatus and Process for Wax Loading of Valves

Exemplary Wax-Deposition Process

[0200]Deposition of wax in valves of the microfluidic network, as at step
1804 of FIG. 18 may be carried out with the exemplary equipment shown in
FIGS. 28A and 28B. The DispenseJet Series DJ-9000 (available from
Asymtek, Carlsbad, Calif.) is a non-contact dispenser suitable for this
purpose that provides rapid delivery and high-precision volumetric
control for various fluids, including surface mount adhesive, underfill,
encapsulants, conformal coating, UV adhesives, and silver epoxy. The
DJ-9000 jets in tight spaces as small as 200 micrometers and creates
fillet wet-out widths as small as 300 micrometers on the dispensed side
of a substrate such as a die. It dispenses fluid either as discrete dots
or a rapid succession of dots to form a 100-micron (4 mil) diameter
stream of fluid from the nozzle. It is fully compatible with other
commercially available dispensing systems such as the Asymtek Century
C-718/C-720, Millennium M-2000, and Axiom X-1000 Series Dispensing
Systems.

[0201]A DJ-9000 is manufactured under quality control standards that aim
to provide precise and reliable performance. Representative
specifications of the apparatus are as follows.

[0203]The DJ-9000 has a normally closed, air-actuated, spring-return
mechanism, which uses momentum transfer principles to expel precise
volumes of material. Pressurized air is regulated by a high-speed
solenoid to retract a needle assembly from the seat. Fluid, fed into the
fluid chamber, flows over the seat. When the air is exhausted, the needle
travels rapidly to the closed position, displacing fluid through the seat
and nozzle in the form of a droplet. Multiple droplets fired in
succession can be used to form larger dispense volumes and lines when
combined with the motion of a dispenser robot.

[0204]The equipment has various adjustable features: The following
features affect performance of the DJ-9000 and are typically adjusted to
fit specific process conditions.

[0205]Fluid Pressure should be set so that fluid fills to the seat, but
should not be influential in pushing the fluid through the seat and
nozzle. In general, higher fluid pressure results in a larger volume of
material jetted.

[0206]The Stroke Adjustment controls the travel distance of the Needle
Assembly. The control is turned counterclockwise to increase needle
assembly travel, or turned clockwise to decrease travel. An increase of
travel distance will often result in a larger volume of material jetted.

[0207]The Solenoid Valve controls the valve operation. When energized, it
allows air in the jet air chamber to compress a spring and thereby raise
the Needle Assembly. When de-energized, the air is released and the
spring forces the piston down so that the needle tip contacts the seat.

[0208]The seat and nozzle geometry are typically the main factors
controlling dispensed material volume. The seat and nozzle size are
determined based on the application and fluid properties. Other
parameters are adjusted in accordance with seat and nozzle choices.
Available seat and nozzle sizes are listed in the table hereinbelow.

[0209]Thermal Control Assembly: Fluid temperature often influences fluid
viscosity and flow characteristics. The DJ-9000 is equipped with a
Thermal Control Assembly that assures a constant fluid temperature.

[0210]Dot and Line Parameters: In addition to the DJ-9000 hardware
configuration and settings, Dot and Line Parameters are set in a software
program (referred to as FmNT) to control the size and quality of dots and
lines dispensed.

Example 7

24-Lane Cartridge

[0211]FIGS. 29A-29C show an exemplary 24-lane cartridge having three
layers in its construction in which there is no hydrophobic membrane, and
no thermally compliant layer. The three layers are a laminate 2922, a
microfluidic substrate 2924, and a label 2926. A typical reaction vol. is
4.5 μl in each lane from 2 rows of 12 lanes. No bubble-removal vents
are utilized and instead of a hydrophobic end vent, there is just a hole.
This is consistent with use of an accurate pipetting system. There is no
thermally compliant/conductive layer for situations where enough pressure
can be reliably applied to the cartridge that effective thermal contact
with the microfluidic substrate can be made without requiring the
additional layer. The absence of two layers from the construction saves
manufacturing costs.

Example 8

96-Lane Cartridge

[0212]FIGS. 30A-D show aspects of a 96-lane cartridge design, including
complementary heater configurations. (FIG. 30A shows cartridge design;
30B shows heater design in a single metal layer; 30C shows individual PCR
channels overlaid with heater configurations; 30D shows individual PCR
lanes.) In the embodiment shown, liquid sample is loaded without air
bubbles as the lanes do not have any vents. Two or more Mux can be
utilized for controlling all 96 PCR channels.

[0213]Such an arrangement lends itself to whole area imaging (e.g., by a
CCD) for detection instead of optical based methods using diodes and
lenses.

Example 9

Real-Time PCR

[0214]FIG. 31 shows a trace of real-time PCR carried out on multiple
samples in parallel with an apparatus and microfluidic network as
described herein. The PCR curves are standard plots that are
representative of fluorescence from 12 different PCR lanes as a function
of cycle number.

[0215]The foregoing description is intended to illustrate various aspects
of the present technology. It is not intended that the examples presented
herein limit the scope of the present technology. The technology now
being fully described, it will be apparent to one of ordinary skill in
the art that many changes and modifications can be made thereto without
departing from the spirit or scope of the appended claims.